Method and device for multi-spectral photonic imaging

ABSTRACT

An imaging device includes (a) a light source device arranged to illuminate a sample under investigation with illumination light, (b) a detector device arranged to collect a plurality of images including at least one sample light image backscattered by the sample, and at least one marker light image originating from at least one marker substance in the sample, and (c) a processor device adapted to process the at least one sample light image.

TECHNICAL FIELD

This disclosure relates to an imaging device, in particular for medicalimaging, like a multi-spectral photonic system, which offerssignificantly better imaging accuracy compared to conventionaltechniques, for surface and sub-surface imaging of a sample including atleast one marker substance. Furthermore, this disclosure relates to animaging method, in particular for medical imaging of a sample includingat least one marker substance, e.g., a human or animal or part thereof.Preferred applications are present in medical imaging and in particularin creating diagnostic images or images used for guiding interventionalprocedures.

BACKGROUND

Photonic imaging is an ideal modality for biomedical diagnostics sinceit relates directly to a physician's vision and offers highly attractivecharacteristics, including use of non-ionizing radiation which does notdamage the tissue, high flexibility in contrast mechanisms, portability,small form factor and real-time image acquisition. Healthy and diseasedtissues exhibit differences in several properties such as structural,compositional, metabolic, molecular and structural. Local or systemicadministration of agents with specificity to cellular and subcellulartissue and disease biomarkers could alter the optical properties ofhealthy and diseased tissue in a different way resulting invisualization of lesions with high contrast with the background healthytissues. Recent studies indicate that the use of externally administeredfluorescent probes is a highly promising approach since fluorescencesignals can provide high contrast. For example, engineered probes can bevery sensitive and specific in cancer detection by targeting specificmolecular features of carcinogenesis and tumor lesions.

The need to efficiently detect the signal from molecular probes led tothe development of several imaging methods and technologies in the lastdecade. Nevertheless, imaging methods used in practice suffer fromlimitations related to a) performance and b) convenience in useespecially in clinical environments.

The imaging performance in resolving superficial fluorescence activitycan be compromised by three major parameters: spatial variation intissue optical properties, depth of the fluorescence activity and tissueauto-fluorescence. The dependence of signal intensity, e.g.,fluorescence, on these parameters can limit both the contrast and theoverall accuracy of uncorrected simple “photographic” or “video”methods. This can be better understood by considering, for example, thata dark, bloody area, significantly attenuates light intensity over aless absorbing region, an effect that can lead to false negatives.Similarly a non-absorbing area may show as probe rich compared to a darkregion even at very moderate amounts of molecular probe. This can leadto false positives. Similar false positives or false negatives can bealso generated as a function of the depth of the fluorescence lesionsince light intensity non-linearly and strongly attenuates as a functionof depth, i.e., light propagation in tissue. Therefore, unless onecorrects for the variation in fluorescence signal intensity due to thevariation of optical properties, variation of depth or auto-fluorescenceraw images of tissue can be inaccurate or contain undesired artifacts.These effects have been noted in the past (e.g., see Ntziachristos et.al. Nature Biotechnology 2005; 23:313-320).

Systems that utilize imaging at multiple wavelengths have been developedto differentiate auto-fluorescence from a fluorochrome of interest.Similarly, variation of the intensity due to tissue optical propertiesand depth is typically corrected in tomographic systems.

On the other hand, further systems that show the potential to overcomethe abovementioned limitations in performance are not suitable forclinical use due to poor functionality characteristics. For example,scanning multispectral systems can provide high spectral resolution butrequire time for scanning and therefore are not suitable for movingobjects, i.e., real-time imaging operation. Therefore, they are notsuitable for use on tissues moving due to breathing or heartbeat.Moreover, information generated by the images is not provided in realtime and therefore such methods are impractical for scanning largetissue areas for lesions, zoom and focus on suspicious areas duringexamination and, last but not least, cannot be used for interventionalprocedures such as real time surgical guidance for lesion excision.

Overall, currently no medical photonic imaging system exists thataccounts for the effects of light propagation and interaction withtissue in real-time to lead to accurate clinical imaging systems, forexample, intra-operative imaging systems.

Tissue lesions, e.g., cancer, exhibit alterations in the tissuemolecular, structural, functional and compositional characteristics. Theuse of targeting probes, e.g., molecular probes, has the potential toprovide significant contrast between healthy and diseased tissue.Especially, with recent advances in genomics, proteomics andnanotechnology, new probes conjugated with appropriate optical markers,e.g., a fluorescent molecule or a photo-absorbing nano-particle, enableeasier and more accurate detection of tissue structural, functional andcompositional properties which could lead to non-invasive in vivodiagnostics. Ideally, an imaging modality able to capture thosedifferences in optical signals and thereby detect and identify tissuelesions in real time could significantly increase our diagnostic,real-time guidance and interventional imaging capabilities.

Although several experimental methods have proven the potential of thisapproach, none of them exhibits sufficient performance for clinical use.The main limitations are: due to high complexity and inhomogeneity ofbiological tissues, photons undergo multiple and complex interactionswith the tissue resulting in alterations to the measured signal.Correction of the measured signals requires a complex model thatcontains aspects of the tissue optical properties and/or geometricalcharacteristics. Reliable measurement of tissue optical propertiesrequires fast acquisition and processing of a large amount ofinformation. Existing imaging methods and technologies are limited as tothe amount of information they can capture and correction they canoffer.

Clinical applications such as surgical guidance require real timediagnostic or pathology feedback. In other words, signal capturing,processing and rendering of diagnostic result should be done in realtime. Existing methods are limited by a tradeoff between analyticalcapabilities and speed.

US 2008/0312540 A1 discloses a system and method providing normalizedfluorescence epi-illumination images and normalized fluorescencetransillumination images for medical imaging. Normalization is obtainedby combining an intrinsic image, like, e.g., a reflection image, and anemitted light image, like, e.g., a fluorescence image, collected at thesample. This conventional technique has limitations in practicalapplications, in particular due to the time needed for collecting imageswith multiple spectral ranges using changing optical filters or filterwheels, the duration of image data processing and a limited imagequality. Furthermore, this technique has a restricted capability ofproviding diagnostic images since it only partially accounts for opticalproperty changes, i.e., it accounts for absorption changes but notscattering changes.

It could therefore be helpful to provide an improved imaging device, inparticular for multi-parametric real-time medical imaging, capable ofavoiding disadvantages of conventional techniques. Furthermore, it couldbe helpful to provide an improved imaging method, in particular forcollecting and providing photonic images for biomedical imaging withimproved accuracy, being capable of avoiding disadvantages ofconventional techniques.

SUMMARY

We provide an imaging device including (a) a light source devicearranged to illuminate a sample under investigation with illuminationlight, (b) a detector device arranged to collect a plurality of imagesincluding at least two sample light images backscattered by the samplein different spectral ranges, and at least one marker light imageoriginating from at least one marker substance in the sample, and (c) aprocessor device adapted to process the at least two sample light imagesand create at least one correction component, the processor devicefurther adapted to correct the marker light image using the at least onecorrection component.

We also provide an imaging method including illuminating a sample underinvestigation with illumination light generated with a light sourcedevice, collecting at least two sample light images from sample lightbackscattered by the sample, collecting at least one marker light imagefrom marker light generated by at least one marker substance in thesample in different spectral ranges, and processing the sample andmarker light images and rendering at least one corrected marker lightimage based on the at least two sample light images and the at least onemarker light image.

We further provide an imaging device including (a) a light source devicearranged to illuminate a sample under investigation with illuminationlight, (b) a detector device arranged to collect a plurality of imagesincluding at least one sample light image backscattered by the sample,and at least one marker light image originating from at least one markersubstance in the sample, and (c) a processor device adapted to processthe at least one sample light image.

We further yet provide a medical imaging device including the imagingdevice including (a) a light source device arranged to illuminate asample under investigation with illumination light, (b) a detectordevice arranged to collect a plurality of images including at least onesample light image backscattered by the sample, and at least one markerlight image originating from at least one marker substance in thesample, and (c) a processor device adapted to process the at least onesample light image.

We also further provide an imaging method including illuminating asample under investigation with illumination light generated with alight source device, collecting at least one sample light image fromsample light backscattered by the sample, collecting at least one markerlight image from marker light generated by at least one marker substancein the sample in different spectral ranges, and processing the sampleand marker light images.

BRIEF DESCRIPTION OF THE DRAWINGS

Further details and advantages are described in the following withreference to the attached drawings.

FIG. 1: a schematic illustration of our imaging device.

FIGS. 2 and 3: schematic illustrations of detectors used in the imagingdevice.

FIG. 4: a schematic illustration of a preferred application of theimaging device.

FIG. 5: a flow chart illustrating steps of the imaging method.

FIG. 6: a schematic illustration of a practical application of thedevice.

FIGS. 7 and 8: photographs illustrating experimental results.

FIG. 9: a schematic illustration of the function of the imaging deviceaccording to one example of the device.

DETAILED DESCRIPTION

We provide an imaging device which comprises a light source devicearranged for illumination of a sample under investigation withillumination light, and a plurality of detectors arranged for collectingmultiple different images of the sample. The detectors include at leastone multi-spectral sample light camera capable of sensing sample lightcoming from the sample, e.g., backscattered (reflected and/orback-emitted) and generated by the sample in different spectral ranges,and collecting at least two sample light images of the sample in thedifferent spectral ranges, and at least one marker light camera capableof simultaneous sensing marker light generated by at least one markersubstance in the sample and collecting marker light images of thesample. The at least two sample light images are used for calculating atleast one correction component. The marker light image is correctedusing the at least one correction component which is a correction imageor another signal including information relevant for correction of themarker light image. Preferably, the sample and marker light images withthe different spectral ranges of light detection are collectedsimultaneously, i.e., at the same time. Time delay as it may occur withthe conventional technique, e.g., with changing optical filters, isavoided. The cameras utilized can be optical cameras includinglight-sensitive chips, e.g., a Charged Coupled Device (CCD) sensor or aCMOS-sensor.

To collect the sample and marker light images simultaneously, ourimaging device includes a light splitting imaging optic which isconfigured for imaging light from the sample onto the detectors. Thesample light is relayed onto the at least one sample light camera, whilethe marker light is relayed onto the at least one marker light camera.Both of the sample light and the marker light are collectedsimultaneously, thus allowing a real time processing of the multipledifferent images of the sample.

Furthermore, our imaging device includes a processor device beingadapted for processing the sample and marker light images in paralleland rendering at least one corrected image based on the at least onemarker light image and the sample light images. Preferentially, the atleast one corrected image is computed and rendered in a real-time mode.Providing at least one corrected image in real-time mode comprises therepresentation of the at least one corrected image on a display at onetime with the image collection or with a delay after the imagecollection such that the delay is negligible in consideration of thetime scale of sample changes or treatment steps.

Providing at least one corrected image in real-time mode also mayinclude providing an image sequence (video sequence) of correctedimages. As an example, the processor device can be configured togenerate a video sequence of the at least one marker light image, atleast one of the sample light images, the at least one corrected image,or the opto-acoustic image or a combination thereof.

We also provide an imaging method which preferably is conducted usingthe imaging device. The imaging method comprises the steps ofilluminating a sample under investigation with illumination lightgenerated with a light source device, collecting sample light imagescreated by sample light backscattered, in particular reflected, by thesample in the different spectral ranges and collecting at least onemarker light image created by marker light generated by at least onemarker substance in the sample, wherein the sample and marker lightimages are collected using a light splitting imaging optic, at least onemulti-spectral sample light camera and at least one marker light camera,and processing the sample and marker light images and rendering at leastone corrected image based on the sample light images and the at leastone marker light image in a real-time mode.

Preferably, the sample is a biological object, in particular a human oranimal body or a part thereof. In particular, the sample comprisesbiological tissue or a part thereof. Accordingly, the device/method ispreferably used for medical imaging.

Generally, the sample light collected with the sample light camera isthe portion of the illumination light reflected (scattered) by thesurface and sub-surface layers of the sample. Accordingly, the samplelight includes contributions of the background body of the sample andpossibly of the at least one marker substance distributed in the sample.The term “sample light image” refers to an incident light image of thesample, like a diffusive reflection image, or a color image, obtained byimaging light at the surface of the sample onto the sample light camera.On the other hand, the marker light refers to light specifically emittedor reflected by the at least one marker substance.

Sensing exclusively the marker light would allow a strict discriminationbetween the sample background light and the marker light and anassignment of the marker light to certain topographic properties of thesample, e.g., to recognize suspicious tissue regions. Thisdiscrimination is difficult due to the broadband characteristics of thesample background and marker substance. With the collection of multiplesample and marker light images in different spectral ranges, thisdiscrimination can be essentially facilitated. As the sample and markerlight image collection is performed simultaneously and the correctedimage is calculated in real-time, an essential advantage is obtained incomparison with conventional medical imaging methods.

Advantageously, we provide a method and a device for photonic medicalimaging of marker substance which can offer accurate, quantitativeimaging of surface and subsurface tissues and tissue markers. Thisperformance is in contradistinction to the current state of the art thatnot offering accurate performance can lead to false negatives and falsepositives. In addition, our methods can enable this accurate performancein real-time collection and processing of multispectral data. Thereal-time system and method is capable of molecular imaging as itassociates with the specific and accurate visualization of administeredmarker substances with specificity to certain healthy and diseasedtissue marker substances.

For the preferred application in medical imaging, we provide three stepsof administration of one or more contrast agents or probes (markersubstances), e.g., molecular probes, multispectral optical imaging,optionally with opto-acoustic imaging, and processing of captured imagesfor real time display of corrected information. The administration stepis provided as an optional feature of the method. It can be omitted ifthe sample already includes the at least one marker substance fornatural reasons or due to a previous treatment. The at least onecorrected image obtained with the imaging method is also calleddiagnostic image. The term “diagnostic image” refers to the fact thatthe image can be used for finding a diagnosis, e.g., by a physicianand/or by a subsequent image evaluation or identify with highspecificity a problematic or suspicious lesion to lead to efficientguidance and intervention, for example, an intervention with therapeuticintend. As an example, the diagnostic image may include a map of thesample highlighting various sample conditions. Similarly, the diagnosticimage can be used to guide surgical intervention or endoscopicallyadministered biopsies and other surgical procedures.

The term “marker substance” refers to any molecule which specificallybinds to a certain target in the sample, like target tissue, targetcells or certain cell components, like proteins, and which exhibits aninteraction with light (UV, VIS and/or IR wavelength ranges) resultingin a specific absorption and/or fluorescence. The concept of use of amarker substance is to highlight one or more tissue characteristicswhich are altered at a presence of a disease. The marker substance isalso called biomarker, probe or contrast agent. It is selected dependingon the binding properties and the spectral properties thereof. Inparticular, the marker substance is selected so that it targets andreveals a molecular, structural, functional or compositional feature ofthe tissue which specifically changes in a gradual manner during thedisease progress. The presence of the marker substance preferably altersthe optical properties of the tissue, e.g., fluorescence or absorbancein a way that the detected optical signal could even reveal the presenceor progress of the disease. The sample includes one or more markersubstances. If multiple different marker substances are provided, theypreferably have different spectroscopic properties.

A disease like cancer is known to cause several tissue alterations andthe probes used as a marker substance are in general designed tohighlight one of those alterations, e.g., metabolic activity.Nevertheless, diseases like cancer do not always express the samecharacteristic changes and therefore, the probes inherently exhibit lowsensitivity. Inversely, in other cases some non-diseased tissues mightalso mimic one of the disease characteristics reducing thereby the probespecificity. Besides cancer, interventions associated with vascular,intravascular, neuronal, cardiac, reconstructive and other diseases andindications are also considered.

With the available specific and sensitive marker substances such asmolecular probes, it is possible to provide an excellent overallperformance and clinical outcome using the method. Moreover, combineduse of multiple probes may be preferred in terms of further increasingthe information obtained for decision making during guidance andintervention or the diagnostic ability since the detection of a diseasecan then be based on several features that characterize the diseaserather than a single feature. Additionally, the combined use of multipleprobes and other chromophoric agents such as non-specific fluorescencedyes could additionally be utilized to correct for potentialinhomogeneities in local blood perfusion which could otherwise affectthe local delivery of the probes and thereby introduce measurements.

Advantageously, the method can be conducted with various types ofimages. Our detectors are adapted to collect at least two image types ofa color image, a fluorescence image, a reflectance image and/or anexcitation image. Preferably, each of the detectors is provided with atleast one camera filter adapted to the image type to be collected.

The marker light is light generated in response to the illumination byat the least one marker substance. Intensity, spectral composition andgeometric distribution of the marker light is determined by the specificinteraction of the illumination light with the at least one markersubstance and the distribution thereof in the sample. Multiple variantsexist for adapting the at least one marker light camera for efficientlysensing the marker light in different spectral ranges. Preferably, theat least one marker light camera comprises two or more camera fieldsbeing sensitive in the different spectral ranges. A light splittingcamera optic is arranged for imaging portions of the light created bythe sample onto the at least two camera fields. Particularly preferredis a variant, wherein the at least one marker light camera is providedfor simultaneous sensing the marker light in at least two differentspectral ranges. In this case an essentially improved specific detectionof the marker substance distribution and discrimination from thebackground sample light can be obtained.

The at least two camera fields may comprise at least two separatelight-sensitive chips, e.g., CCD chips and/or at least two independentlysensitive areas within one common light-sensitive chip, e.g., CCD chip.This allows a flexible adaptation of the imaging device to therequirements of a practical application where different signalscollected utilize different signal strengths and different dynamicranges. Preferably, the sensitivity of one or more of the independentlylight-sensitive field is automatically adapted, through variableattenuation or amplification of the sample signals collected or thecorresponding electrical signals generated. Preferably, each of the atleast two camera fields is provided with a field filter being adjustinga spectral sensitivity range of the respective camera field. Ifchangeable field filters are provided, the flexibility even can beimproved.

Advantageously, the illumination light can be designed in terms ofgeometric, temporal and/or spectral properties for adapting theillumination light to sample properties (including marker substanceproperties) and image types to be collected. To this end, the lightsource device preferably includes at least one illumination lightconditioning device adjusting at least one of a spectral characteristic,a temporal characteristic, a polarization, a direction and a light fieldshape of the illumination light. If the illumination device includesmultiple light sources, for each of them a specific illumination lightconditioning device can be provided. Preferably, the illumination lightconditioning device comprises at least one of a spectral filter, apolarization filter, an illumination optic and a bundle of opticalfibers.

Preferably, the optical imaging can be combined with opto-acousticsensing. Opto-acoustic is a promising modality that combines advantagesof optical imaging, i.e., high optical contrast mechanisms andtomographic methods, i.e., high penetration depth. The combination ofoptical and optoacoustic sensing is ideal since both modalities canutilize the same marker substances. Therefore, an opto-acoustic imagingdevice is arranged for collecting a multi-spectral opto-acoustic imageof the sample. The opto-acoustic imaging device is capable of collectingat least opto-acoustic data, but preferably an opto-acoustic image ofthe sample.

Accordingly, our device can be provided as an opto-acoustic device,offering real-time collection and correction capability, i.e., as acombination of the optical, CCD-based device and the opto-acousticdevice. In this case, the CCD-based device used for guidance and largefield of view detection and the opto-acoustic device used for resolvingwith high resolution contrast from suspicious regions found on theCCD-based device. A similar practice can also utilize a portableconfocal or multi-photon microscope imaging system.

The processor device of the imaging device is an important featureproviding the capability of real time imaging of the sample, where theimages produced in real time in particular are corrected images thataccount for features that can lead to artifacts (i.e., false positive orfalse negative readings) that may be present in the raw images.Preferably, the processor device includes at least one of FieldProgrammable Gate Arrays and Graphic Processing Units. As an advantage,those types of processors are commercially available at relatively lowprices and provide the ideal solution for dedicated real time dataprocessing.

Optionally, a control device can be provided additionally to theprocessor device. The control device can be configured for at least oneof the following functions. Firstly, it can control the light sourcedevice, the detectors and/or the processor device. Secondly, the controldevice can be provided with a display device displaying the at least onesample light image, at least one of the marker light images, the atleast one corrected image and/or the opto-acoustic image. Finally, thecontrol device may be connected with an administering device whichconfigured for introducing the at least one predetermined markersubstance into the sample. The processor device and the control devicecan be implemented within a common computer unit.

The flexibility of the imaging device can be further improved if thedetectors are arranged with a modular structure having a plurality ofcouplings each of which being arranged for accommodating one of thedetectors. Advantageously, this allows a simple adaptation of theimaging device to the requirements of a particular application.

Further preferably, the step of rendering the at least one correctedimage includes an image data correction procedure. “Image datacorrection” refers to preferentially and independently modifying theinformation contained to each pixel of a “final image” also presented tothe systems operator so that predetermined features of the image areimproved and convey to the operator more accurate information.

The image data correction applied is based on the multi-parametric datacollected in real-time, and may also contain a-priori knowledge, in theform of information stored in the image processing device prior to thereal-time measurement. Correspondingly “image data correction” alsorefers to change the intensity in the pixels of the image corrected andsubsequently projected so that the resulting image more accuratelyreflects the actual marker bio-distribution in the field of view of theimage. The image data correction therefore may contain any of steps thatimprove the intensity in each image 1) from the effects of contaminationof marker signals with endogenous tissue signals such asauto-fluorescence signals, 2) from the effects that the depth of themarker location has on the marker signal collected, and/or 3) the effectthat the tissue optical properties have on the marker signal collected.For example, the effect that a strong absorber co-localized with amarker fluorochrome has on the intensity recorder from the markerfluorochrome. Particular examples of image data correction are describedbelow.

Preferred example are described in the following with particularreference to the optical and opto-acoustic set-up and data processingstructure provided for obtaining multispectral photonic images in realtime. Details of selecting suitable marker substances, preparing asample, like introducing at least one marker substance type into thesample, designing the imaging optics, in particular with regard tofocusing and magnification properties, operating the at least one lightsource, detectors and optionally further sensors, like an opto-acousticsensor and image processing techniques are not described here as far asthey are known from prior art, in particular from conventional photonicsystems for medical imaging. Furthermore, the imaging devices presentedin the figures can be implemented in different ways depending on thespecific needs of an application.

FIG. 1 illustrates features of preferred example of the imaging device100 which comprises a light source device 10, a detector device 20 withdetectors 21, 22, a light splitting imaging optic 30 and a processordevice 40. Optionally, additionally a control device 50, anopto-acoustic sensor device 60 and/or an administering device 70 can beprovided. The imaging device 100 is configured to image a sample 1 withthe imaging method. The sample 1 is, e.g., a biological sample, like ahuman or animal body or a part thereof. For in vitro investigations, thesample 1 is arranged on a carrier, while with an in vivo investigation(see FIG. 4) the sample 1 is a region of investigation included in thebody of the proband (patient).

The light source device 10 comprises two light sources 11.1, 11.2, anoptical filter 12 and light focusing and homogenizing units 13.1, 13.2,which are arranged to illuminate the sample 1 with illumination light.The components 12 and 13.1 being connected via an optical fiber 14 andthe component 13.2 provide illumination light conditioning devices,which are adapted for adjusting the spectral characteristic (inparticular with the optical filter 12) and the polarization, directionand/or light field shape of the illumination light (in particular withthe light focusing and homogenizing units 13.1, 13.2). Additionally oralternatively, the illumination light conditioning device may beprovided with a temporal illumination control, like a shutter, aswitching device or a light source control (not shown).

While the method can be implemented with a single light source, like,e.g., a laser source, the light source device 10 preferably comprisesmultiple light sources, which provide advantages in terms of providingillumination light with predetermined spectral and temporalcharacteristics. As an example, the light source device may comprise atleast one broadband light source 11.1, e.g., a white light tungstenbulb, a halogen lamp or a broadband LED, or at least one narrowbandlight source 11.2, like, e.g., a narrowband laser or LED. When multiplenarrowband light sources are utilized, the different bands can beemployed to excite at least one marker substance in tissue, which can beemployed to visualize multiple markers simultaneously or correct for theeffects of depth, since different spectral bands can probe differentdepths. With the provision of multiple light sources, the illuminationlight conditioning device comprises multiple filters, focusing andhomogenizing components. In terms of temporal characteristics, the lightsource can be continuous (CW), pulsed or intensity modulated. The lightfrom each light source 11.1, 11.2 could be delivered from each sourcedirectly to the sample or the output of all light sources could becombined and delivered by a single optical arrangement, e.g., fiberoptic bundles or lens.

The light from the sources, each one separately or all together could befiltered to achieve the necessary spectral characteristics. As anexample, IR light might be filtered out from white light indented forcolor imaging so that there is no crosstalk with the IR fluorescencesignal detection. Moreover, the use of polarizers at the illuminationand imaging optical paths could minimize effects of specular reflection.

The detector device 20 comprises three cameras 21, 22 arranged tocollect sample light and marker light, respectively. The sample lightand the marker light is relayed from the sample 1 to the cameras 21, 22using the light splitting imaging optic 30, which comprises an imagingoptic 31 collecting the light from the sample 1, multiple opticalfilters 32, 34, 35 being arranged to adjust spectral features of thesample light and marker light, and an image splitter 33 separating thelight paths from the imaging optic 31 towards the cameras 21, 22. Theoptical filters 32, 34, 35 (camera filter) can comprise filter wheelsloaded with appropriate band pass filters, which enable the selection ofthe imaging spectral band for each camera independently. The imagesplitter 33 comprises two semi-transparent plane mirrors 33.1, 33.2,wherein, as an example, the first mirror 33.1 is a dichroic mirror thatreflects visible light towards the sample light camera 21 and has 10%reflectance and 90% transmittance, while the second mirror 33.2 has 5%reflectance and 95% transmittance.

The sample light camera 21 is arranged to collect the sample light imageof the sample 1. The spectral characteristic of the sample light isadjusted with the optical filter 34, which, e.g., suppresses spectralranges of marker substance fluorescence or passes visible light. Thesample light camera 21 includes a camera sensor, like, e.g., a CCDsensor as known from conventional photographic cameras. The sample lightcamera 21 is connected with the processor device 40, wherein the samplelight image is processed (see below).

The cameras 22 have a more complex structure as they are arranged forsensing marker light in different spectral ranges simultaneously in realtime. To this end, the marker light cameras 22 have a structure asfurther illustrated in FIGS. 2 and 3. The marker light cameras 22 areconnected with the processor device 40, wherein the marker light imagesare processed in parallel together with the sample light image to renderat least one corrected image in real time. Details of image processingare discussed below with reference to FIGS. 5 and 6. Alternatively, oneof the cameras in 22 may be arranged to detect a third light such asauto-fluorescence, or a sample light, at one or more spectral bands, asdescribed in FIG. 1 and FIG. 3, giving flexibility in the utilization ofthe proposed example.

The light splitting imaging optic 30 is arranged in a casing (not shown)shielding the light paths from the sample 1 to the cameras 21, 22. As anexample, the casing can be structured with a tube shape as it is knownfrom microscopes. The detectors 21, 22 are connected with the casinghaving a modular structure with a plurality of couplings each of whichbeing arranged for accommodating one of the detectors.

FIG. 2 schematically illustrates a sectional view of a marker lightcamera 22, which comprises at least two camera fields 23, 24 each with afield filter 27. Furthermore, the marker light camera 22 comprises alight splitting camera optic 25, which is adapted to split marker lightrelayed from the light splitting imaging optic 30 (see FIG. 1) towardsthe camera fields 23, 24. On each of the camera fields 23, 24, acomplete image of the sample (or region of interest) is created. Thelight splitting camera optic 25 comprises a combination of mirrorsand/or prisms as it is known from conventional image splitters.

The camera fields may comprise separate CCD chips 23 as schematicallyillustrated in FIG. 3A and/or sensitive areas 24 of one common CCD chip26 as illustrated in FIG. 3B. In the first case, each of the CCD chips23 is connected with the processor device 40 for transferring the markerlight image data. In the second case, the common CCD chip 26 isconnected with the processor device 40, wherein image data belonging tothe different sensitive areas 24 are separated with a data processing inthe processor device 40.

The processor device 40 (FIG. 1) is configured to process the sample andmarker light images in parallel and rendering at least one correctedimage based on the at least one marker light image and the sample lightimages in a real-time mode. Real time processing and rendering of theimaging data is particularly important for the clinical use of adiagnostic system. Nevertheless, this real time processing is quitedemanding and conventional computer CPUs may have an insufficientperformance. Therefore, preferably, the processor device 40 has aseparate processor dedicated to image data processing. Such processorscould be Field Programmable Gate Arrays (FPGAs) and Graphics ProcessingUnits (GPUs).

According to FIG. 1, the control device 50 is connected with theprocessor device 40 and with the cameras 21, 22. The connection with thecameras 21, 22 can be provided directly or via the processor device 40.Furthermore, the control device 50 is connected with the light sourcedevice 10, the opto-acoustic sensor device 60 and the administeringdevice 70. Thus, the control device 50 is capable for controlling thecomplete operation of the components of the imaging device 100. Thecontrol device 50 is provided with a display 51 illustrating operationconditions of the imaging device 100 and/or images collected with thecamera device 20 and/or calculated with the processor device 40.

The opto-acoustic sensor device 60, including, e.g., an ultrasonicarray, is arranged to collect an opto-acoustic image of sample 1simultaneously or guided by the collection of the sample and markerlight images. The opto-acoustic modality could provide complementaryinformation, e.g., about the lesion morphology in deeper layers of thetissue or resolve the same marker with depth resolution and as afunction of depth, in particular when using multi-spectral optoacoustictomography (MSOT). To this end, the opto-acoustic sensor device 60 isconfigured to subject the sample 1 to one or multiple excitation lightpulses and for collect a mechanical wave response created in the sample1 as it is known from conventional opto-acoustics. The excitation lightpulse can be created with one of the light sources 11.2 or, in otherwords, one light source 11.2 of the light source device 10 can beintegrated in the opto-acoustic sensor device 60.

The imaging system of FIG. 1 facilitates an optical construction thatsplits the light collected by the imaging optic 31 into multiple imagingchannels. The light splitting is performed such that each of the imagingchannels measures one or more spectral bands of fluorescence and/orreflectance light. This light splitting example is designed to minimizethe crosstalk between the imaging channels and maximize the opticalthroughput.

Each imaging channel utilizes additional optical filtering (filters 34,35) to ensure the purity of the measured signal. Each of the cameras 21,22 can be conventional monochrome or color, multispectral, time-resolvedor intensity modulated. The light splitting example can optionallyutilize one or more relay lenses to form an image of the correct sizeand magnification on each of the camera sensors. Light splitting can beperformed by any combination of partially reflecting mirrors or prisms,dichroic or polichroic mirrors and polarization splitters. For theimaging optic 31 can be used any imaging example that can collect lightand form an image such as refractive and/or reflective (catoptrics)elements.

In a practical example, the imaging device 100 of FIG. 1 is configuredfor intraoperative fluorescence imaging. It enables simultaneous imagecapturing of color, fluorescence and intrinsic (excitation spectralband) imaging for marker substances consisting of IR-fluorescent probes,e.g., Cy5.5 and AlexaFluor750. For this application, the sample lightcamera 21 is a color camera coupled with a filter for the visiblewavelength range to ensure only visible photons will be detected. Thehalogen lamp light source 11.1 with the infrared light filtered out isused for white light illumination of the sample 1 for the purpose ofcolor imaging. The second light source 11.2 is a laser for theexcitation of fluorophores. The laser can be a diode laser at thewavelength of 673 nm for the excitation of Cy5.5-labeled probes or a 750nm for the excitation of AlexaFluor750-labeled probes. For the whitelight source 11.1 and the laser 11.2, the light is delivered to thesample 1 (e.g., tissue) through a fiber optic bundle and a multimodefiber respectively to a collimator and a diffuser (13.1, 13.2) for beamexpansion and uniform illumination. Alternatively, one of the cameras 22collects intrinsic tissue fluorescence, or fluorescence coming fromtissue at another spectral band than the one that the first fluorescencecamera is operating in so that it can derive and correct forauto-fluorescence or measure a second target marker fluorochrome orintrinsic tissue fluorochrome or chromophore, for example, at least oneform of hemoglobin.

Light from the sample 1 under examination is collected using a zoom lensof the imaging optic 31. Alternative to the zoom lens could be used anyoptical imaging system such as endoscope or microscope. Two linearpolarizers with perpendicular polarization axes can also be employed atillumination and imaging light paths to eliminate specular reflection.The primary image of the sample 1 formed by the zoom lens falls at thefocal plane of each relay lens group of three imaging channels.

FIG. 1 is a schematic representation of the imaging platform. Theimaging device 100 of FIG. 1 is capable of capturing any combination ofthe following:

-   -   color image so that a surgeon will be able to recognize an area        under examination,    -   multispectral fluorescence images,    -   multispectral reflectance images, and    -   opto-acoustic signals.        All this optical information captured will be used to calculate        images of diagnostic value as outlined below.

In this example, the imaging device 100 employs three imaging channelsbut in the exact same way it could be implemented with less (two) ormore imaging channels, as shown in FIG. 6. Intrinsic images in atwo-camera configuration can be provided by processing the color imageto convert it to a near-infrared attenuation map, based on spectralinformation relating visible and near-infrared spectra already stored inthe processing device. Furthermore, while features are described abovewith exemplary reference to an optical set-up, which has a structuresimilar to an optical microscope, it is emphasized that theimplementation is not restricted to this illustrated structure, butrather possible with more compact apparatuses. In particular, theimaging optics, image splitting optics and cameras can be integratedwithin one single compact casing or within two casings as illustrated inFIG. 4. It is even possible to miniaturize the imaging device such thatit can be used for conventional medical imaging techniques, like, e.g.,endoscopy.

FIG. 4 shows application of the technique for medical imaging, whereinthe sample 1 is a part of a human 2. The sample 1 (region ofinvestigation) is, e.g., a surface region or a sub-surface tissueregion, like a subcutaneous tissue region imaged during a surgicaloperation and/or using an endoscopy instrument. The imaging device 100comprises the illumination device 10, the light splitting imaging optic30, the detector device 20 and the processor and control device 40, 50.Reference numeral 71 indicates parts of an administering device 70,which is adapted to introduce at least one marker substance to thesample 1 and which includes a marker substance reservoir, a supplyconduit (like a syringe needle) and a drive unit. The administeringdevice 70 can be adapted for an automatic operation controlled by thecontrol device 50 (see FIG. 1).

FIG. 4 illustrates that the method fundamentally consists of threeparts, namely 1) an administration of a diagnostic probe (including theat least one marker substance, in particular at least one fluorescenceagent), either systemically (3, 4) or locally (5), 2) operating theimaging system, and 3) real time processing and displaying. Thus, anext-generation intra-operative imaging platform based on real-timemulti-spectral image capturing, processing and rendering combined withthe use of fluorescence agents is provided. Further details aredescribed in the following with reference to FIGS. 5 and 6.

FIG. 5 illustrates the steps of the imaging method from data acquisitionuntil calculation of a diagnostic image, comprising preparing the samplewith at least one marker substance (S0), data acquisition (includingmultispectral optical imaging and multispectral opto-acoustic imaging)resulting in raw image data (S1), data correction (including correctionfor overlapping fluorescence spectra, overlapping absorption spectra, ortissue optical properties such as absorption, scattering, and depthdistribution of marker substances) resulting in corrected images ofmarker substance distribution, like a fluorophore and/or chromophoredistribution (S2), an image processing, in particular image calculation(including a combined assessment of multiple marker substances,assessment of temporal characteristics of image signals and/orhighlighting particular areas or patterns in a color image) resulting ina diagnostic image (S3), and a further processing of the diagnosticimage, like, e.g., image processing, storing, printing, displaying,recording or the like (S4). Steps S1 to S3 represent the essentialreal-time steps of the imaging method, while steps S0 and/or S4represent optional features, which can be omitted or conducted at timesdifferent from the time of imaging. As an example, the imaging methodcan be applied with a sample, which already includes the at least onemarker substance for natural reasons or due to a preparation in thepast.

In more detail, the calculation of images having a diagnostic value,based, e.g., on the color image, the multispectral fluorescence images,the multispectral reflectance images, and opto-acoustic signals can bedone conceptually in two stages (see FIG. 5):

-   -   (A) Calculate artifact-free fluorescence and reflectance images        which represent the spatial distribution of fluorophores and        chromophores (step S2).    -   (B) Use the fluorescence and reflectance images to calculate a        diagnostic map of the tissue, i.e., identifying the healthy and        diseased tissue (step S3).        (A) Data Correction

Correction procedures applied in real-time are a preferred feature asthey lead to artifact-free marker images (i.e., fluorescence images)which accurately represent the spatial distribution of fluorophores orchromophores. Several corrected schemes can be responsible for anintegrated correction of all effects that may undergo in a real-timemeasurement, although a selective number of steps may be applied atspecific applications. The generic model considered herein is that thepixel intensity P(x,y) in a raw image is a function of multiplecontributions, i.e.,P(x,y)=f(Pr(x,y), m(x,y,d(x,y)), q(x,y), d(x,y)),  Eq. 1,whereby, d(x,y) represents the depths of the marker accumulation, q(x,y)is the contribution of intrinsic signals due to tissue intrinsicfluorochromes and chromophores, m(x,y) is the contribution to the signalof the optical properties in tissue, in particular the attenuation fromabsorption and scattering, which is also a function of depth and Pr(x,y)is the “real” signal, i.e., the artifact free marker image that is ofinterest.

In a typical form, Eq. 1 is not linear and solutions can be found withminimizations. However, under certain assumptions, Eq. 1 can becomelinear, with each term linearly contributing to the raw image. In thiscase of linear dependence the solution for the “real” image Pr(x,y) canbe simply written as:Pr(x,y)=P(x,y)−m(x,y,d(x,y))−q(x,y)−F(d(x,y)),  Eq. 2whereas, F(d(x,y)) herein is a generic function that can correct alsofor the effects for depth on the signal. The overall correction wouldalso be based on a regression algorithm that identifies the uniquespectral components of the “marker” over the contributions or signalalternate from the other contributions. In the following, preferredprocessing schemes are discussed as implementation examples:1. Correction of Optical Properties

Variation in light attenuation in tissues can contribute to modifyingthe signal recorded from the marker. To correct such effects the methodteaches of processes that improve on such artifacts. The essence ofcorrection is to independently capture the variation of opticalproperties and correct the marker signal based on this variation. Anexample is that varying absorption due to haemoglobin preferentiallyattenuates fluorescence signals at areas of high absorption, which canlead to artifacts if not corrected.

-   -   a) Preferably, multi-spectral measurements are applied to        capture the spectral variations of optical attenuation of the        illumination, as it appears on the intrinsic (sample light)        image. These images can be unmixed in real time to reveal the        haemoglobin concentration (by unmixing the oxy- and deoxy-known        spectra) and also to reveal scattering by simultaneously fitting        for the 1/λ^(a) dependence of tissue scattering, where λ is the        wavelength of measurement and a is a factor that is determined        experimentally. Alternatively a time-resolved or frequency        resolved camera can be employed for “intrinsic measurements” to        independently characterize tissue absorption or scattering.    -   b) In an alternative example, correction is based on utilizing        or adjusting previously known optical measurements by        classifying tissue types as identified on the intrinsic images        and using this allocation of optical properties to similarly        correct the images.

In either case, image correction is based on Eq. 1, the most simplisticform in this case, applied only for corrections for optical propertieswould take the form:Pr(x,y)=P(x,y)*F(μ_(s)′(x,y), μ_(a)(x,y))/Pi(x,y)  Eq. 3or more simplyPr(x,y)=P(x,y)/g*μ _(s)′(x,y)*Pi(x,y)  Eq. 4whereby μ_(s)′(x,y), μ_(a)(x,y) is the reduced scattering coefficientand absorption coefficient respectively of tissue at each image pixeland Pi(x,y) is an attenuation image (intrinsic image) measured at aspectral region that is identical or close to the one utilized by thefluorescence measurements. In this case Eq. 4 makes use of the fact thatthe ratio of fluorescence to intrinsic tissue is relatively insensitiveto absorption but depends by a factor ¾π to the reduced scatteringcoefficient.

Finally the correction for optical properties can occur simultaneouslywith correction for depth as described in the following paragraph.

2. Correction for Optical Properties and Depth

When the fluorophore is covered by a thin layer of tissue, then thefluorescence signal depends on the optical properties of the overlayingtissue in a dual way:

-   -   (a) the excitation light, typically monochromatic, is attenuated        by absorption and scattering so that only a portion of light        reaches the fluorescence marker and induces fluorescence, and    -   (b) the emitted fluorescence is also attenuated while        propagating through the tissue. The fluorescence light,        typically broadband, undergoes different degree of attenuation        for every wavelength depending on the absorption and scattering        properties of the tissue.

Nevertheless, absorption in tissue is predominately due to hemoglobin(oxy- and deoxy-) which have specific, well known absorption spectra.Similarly, tissue scattering spectrum has relatively small variations inshape. Therefore, when the fluorescence light diffuses through a thinlayer of tissue, its spectrum is altered in a characteristic waydepending on the thickness and the concentration of absorbers andscatterers within the overlaying tissue layer. Multispectral measurementof the fluorescence signal can reveal these spectral alterations andcorrect them.

The measured fluorescence signal, i.e., S, is a function of someparameters:S(x,y,λ)=F(C _(m)(x,y,d),OP _(m)(λ),C _(k)(x,y,d),OP _(k)(λ),I_(ex)(x,y,d))  Eq. 5whereby C_(m)(x,y,d) and OP_(m)(λ) are the concentration and the opticalproperties of the fluorescence marker respectively. C_(k)(x,y,d) andOP_(i)(λ) are the concentration and the optical properties of tissuechromophores, fluorophores and scatterers that consist the tissue layer,e.g., k=3 oxy-, deoxy-hemoglobin and total tissue scatterers, d is thedepth of the fluorescence marker or equivalently the thickness of theoverlaying tissue and consequently, I_(ex)(x,y,d) is the intensity ofexcitation light reaching the fluorescence marker at depth d and dependson tissue optical properties:I _(ex)(x,y,d)=I ₀ *G(C _(k)(x,y,d),OP _(k)(λ))  Eq. 6whereby, I₀ is the intensity of excitation light illuminating the tissueunder examination.

The optical properties, i.e., absorption, scattering and fluorescencespectra of the tissue components and the fluorescence marker, OP_(i)(λ)and OP_(m)(λ), are known and can be used as priors. Measurements of thefluorescence signal at multiple wavelengths (λ_(i), i=1, 2, . . . n)will result in a system of n equations (Eq. 5) where n≥k+1. The solutionof this system of equations results in the actual concentration of thefluorescence marker C_(m)(x,y,d) but also the optical properties of thetissue layer, i.e., C_(k)(x,y,d). Thereby, the fluorescence signaloriginating from sub-superficial layer is corrected for tissue opticalproperties and depth of the marker.

3. Overlapping of Fluorescence Spectra/Auto-Fluorescence Correction

To enable simultaneous measurement of multiple probes, i.e., markermolecules that mark multiple targets, and also for retrievingpotentially wanted or unwanted signal contributions coming frombackground intrinsic molecules (i.e., intrinsic chromophores,fluorochromes and the like) background it is a common practice toperform spectral un-mixing of known or potentially even unknowncontributions. Due to the general wide spectral contributions of tissueand external fluorochromes there may be significant spectral overlappingthat cannot be otherwise resolved unless with spectral unmixingtechniques (blind or deterministic) or similarly linear regression formatch the spectral measurements measured to the assumed backgroundcontributions.

(B) Image Calculation

Although, artifact free fluorescence and reflectance images canaccurately provide the spatial distribution of fluorescence probes andtissue chromophores, it is not always straight forward to conclude if atissue area is diseased or not. The diagnostic capability of eachdiagnostic probe is based on targeting a characteristic difference ofthe disease compared to healthy tissue. Nevertheless, diseases likecancer exhibit a remarkable variability in their characteristics andtherefore, a single probe targeting a single cancer feature is often notvery specific. By combined evaluation of multiple cancer features bymultiple probes, can provide significantly increased specificity andsensitivity. Additionally, automatic fusion of the diagnosticinformation with the color image of the tissue under examinationprovides a convenient and easy to use modality, suitable for clinicaluse.

FIG. 6 illustrates another example of a two channel imaging device 100for intraoperative fluorescence imaging. The imaging device 100 inparticular comprises a light source 11, e.g., a halogen lamp, and anotch filter 12 to illuminate a sample 1; a light splitting imagingoptic 30 with an imaging optic 31.1, e.g., a zoom lens, relay lenses31.2, and an image splitter 33, e.g., a plate beam splitter with 10%reflectance and 90% transmittance; a sample light camera 21 having avisible light filter 34; and a marker light camera having a narrow bandpass filter 35.

The system of FIG. 6 is capable of measuring simultaneously, color,fluorescence and intrinsic (excitation wavelength) images for markersubstances (fluorescence probes) emitting in the near infrared region.Nevertheless, if the emission spectrum of the fluorescence probe fallswell within the visible range, e.g., FITC, then the simultaneousmeasurement of fluorescence and reflectance is not possible with thesame configuration.

FIG. 6 further illustrates a method that allows simultaneous capturingcolor reflectance image and visible fluorescence. This method is basedon the combination of the notch filter 12 and the complimentary narrowband pass filter 35 of the marker light camera 22. The notch filter 12blocks a narrow spectral band and transmits the rest. In this setup, thelight from the white light source 11 is filtered using a 532 nm notchfilter. Thereby, the same light source can be used for both white lightillumination and fluorescence excitation. Light collected by the imagingoptic 31 is split in two imaging channels, for visible and fluorescencemeasurements. The fluorescence channel employs a complementary to thenotch filter, narrow band pass filter 35. Thereby, only the fluorescencelight reaches the marker light camera 22 as all the reflected light isrejected by the band pass filter. In analogy to the precedingparagraphs, this implementation correction for optical properties comesfrom processing the at least two spectral bands provided by camera 21 (acolor camera in a preferred example).

FIG. 7 shows images (photographs) captured using the imaging device ofFIG. 6. FIG. 7a shows the color image collected with the sample lightcamera 21, FIG. 7b shows the fluorescence image at 532 nm collected withthe marker light camera 22, and FIG. 7c shows a pseudocolor imageresulted from a fusion of images 7 a and 7 b. Even with theblack-and-white-representation presented here for technical reasons, theessential advantage of the fused image 7 c over the color image 7 a isclearly demonstrated.

FIG. 8 shows images of a mouse with intra-peritoneal tumors. FIG. 8ashows a reflectance color images which illustrates the anatomicalfeatures of the animal's body. FIG. 8b shows a bioluminescence imagewhich reveals the tumor spots. The bioluminescence image is used here asa reference image which accurately reveals the exact tumor location andsize. FIG. 8c shows a fluorescence image at 716 nm originating from adiagnostic fluorescent marker injected prior to imaging session. Rawfluorescence signal does not illustrate one of the tumor spots at theupper-right part of the image, indicated with a dashed arrow, and alsodoes not reveal correctly the size of a second spot at the lower-rightpart of the image, indicated with an arrow. FIG. 8d shows a correctedfluorescence image where all the tumor spots are illustrated correctly.The correction of the fluorescence image (marker light signal) wasperformed using the reflectance spectral images (sample light signals)which are not shown in FIG. 8.

FIG. 9 shows a schematic of the function of an example of the imagingdevice.

According to FIG. 9, the detector may include a combination of opticaland opto-acoustic sensors and detects signals originating from theexamined sample. The output of the detector is at least 3 images whichare:

-   -   at least two images of sample light, captured at different        spectral bands, and    -   at least one image of marker light (e.g., fluorescence).

The images captured by the detector, are processed by a processor. Theprocessing can be done in two stages:

-   -   a. the at least two sample light images are processed to produce        at least two correction components, e.g., correction images.        These correction components could relate to the optical        properties of the sample, e.g., absorption and scattering.    -   b. the at least one marker light image, e.g., fluorescence        image, is processed using at least the two correction components        calculated in process (a). This process calculates at least one        corrected marker light image.

The processor could also perform other processes like combining the twocorrection components into one, which is then used to correct the markerlight image or utilize opto-acoustic data to yield further informationon absorption for more accurate correction. The output of the processoris at least one corrected marker light image.

The features disclosed in the above description, the drawings and theclaims can be of significance both individually as well as incombination for the realization of the device and method and variationsthereof.

This is a continuation of U.S. patent application Ser. No. 13/578,414,filed Aug. 10, 2012, which is a § 371 of International Application No.PCT/EP2010/006937, with an international filing date of Nov. 15, 2010(WO 2011/098101 A1, published Aug. 18, 2011), which is based on EuropeanPatent Application No. 10001478.6, filed Feb. 12, 2010, and U.S. PatentApplication No. 61/304,008, filed Feb. 12, 2010, the subject matter ofwhich is incorporated by reference.

The invention claimed is:
 1. An imaging device comprising: (a) a lightsource device arranged to illuminate a sample under investigation withillumination light, said light source device including a narrowbandlight source arranged to excite a marker fluorescence from at least onemarker substance in the sample and a broadband light source arranged toilluminate the sample, (b) a detector device including at least onemulti-spectral sample light camera and at least one marker light cameraand being arranged to collect a plurality of images in response to saidillumination with the narrowband light source and the broadband lightsource, said plurality of images including: at least one sample lightimage, reflected by the sample in at least one spectral range, and atleast one marker light image originating from at least one markersubstance in the sample, wherein the at least one sample light camera isprovided with an optical filter which is arranged for passing visiblelight for adjusting the spectral range of the sample light reflected bythe sample, wherein a light splitting imaging optic is provided which isconfigured for imaging light from the sample onto the cameras byrelaying sample light onto the at least one sample light camera andmarker light onto the at least one marker light camera so that both ofthe sample light and the marker light are collected simultaneously, and(c) a processor adapted to process the at least one sample light image,and create at least one correction component from the at least onesample light image, the processor device further adapted to correct themarker light image using the at least one correction component.
 2. Theimaging device according to claim 1, wherein the processor deviceprocesses the sample and marker light image and renders at least oneprocessed image in real-time.
 3. The imaging device according to claim1, wherein the processor device calculates the at least one correctioncomponent from the sample light images captured in the red, green andblue regions of the spectrum.
 4. The imaging device according to claim1, further comprising a light splitting imaging optic configured toimage light from the sample onto cameras of the detector device byrelaying sample light onto at least one sample light camera and markerlight onto at least one marker light camera so that both of the samplelight and the marker light are collected simultaneously.
 5. The imagingdevice according to claim 1, wherein the processor device processes theat least the one sample light image to produce a correction componentwhich is then used to correct the marker light image and render at leastone corrected marker light image in a real-time mode.
 6. The imagingdevice according to claim 1, wherein the processor device processes theat least one sample light image to produce at least one correctioncomponent primarily associated with absorption properties of the sampleand at least one correction component primarily associated withscattering properties of the sample.
 7. The imaging device according toclaim 1, wherein the processor device performs correction of the markerlight image using any combination of correction components: a correctioncomponent primarily associated with absorption properties of the sample,a correction component primarily associated with scattering propertiesof the sample, a correction component primarily associated withautofluorescence of the sample, a correction component primarilyassociated with spatial characteristics of illumination, and acorrection component primarily associated with depth distribution of themarker.
 8. The imaging device according to claim 1, wherein: the sampleand marker light images are collected with at least two imagingchannels, and each imaging channel includes at least one optical filterand at least one imaging detector.
 9. The imaging device according toclaim 1, wherein the light source device includes at least oneillumination light conditioning device that adjusts at least one of aspectral characteristic, a temporal characteristic, a polarization, adirection and a light field shape of the illumination light.
 10. Theimaging device according to claim 9, wherein the at least oneillumination light conditioning device comprises at least one of aspectral filter, a polarization filter, an illumination optic and abundle of optical fibers.
 11. The imaging device according to claim 1,further comprising an opto-acoustic imaging device arranged to collectan opto-acoustic image of the sample.
 12. The imaging device accordingto claim 11, wherein the processor device uses the opto-acoustic imagefor at least one of: processing the opto-acoustic image to produce atleast one additional correction image which is then used for thecorrection of the marker light image, displaying the opto-acoustic imageof the sample, and reassembling the opto-acoustic image with thecorrected optical image into one image and rendering that image in areal-time mode.
 13. The imaging device according to claim 1, furthercomprising a computer including at least one of: the computer isconfigured to control at least one of the light source device, thedetectors and the processor device in real time, the computer connectsto a display device displaying the at least one of the at least onesample light image, marker light image, correction image, correctedmarker light image, opto-acoustic image or a combination thereof in realtime, the computer connects to an administering device configured tointroduce at least one predetermined marker substance into the sample.14. The imaging device according to claim 1, wherein the processordevice includes at least one of Field Programmable Gate Arrays andGraphic Processing Units.
 15. The imaging device according to claim 1,wherein detectors of the detector device are arranged with a modularstructure having a plurality of couplings each of which is arranged toaccommodate one of the detectors.
 16. An imaging method utilizing animaging device, the method comprising: illuminating a sample underinvestigation with illumination light generated with a light sourcedevice, collecting at least one sample light image from sample lightreflected by the sample, collecting at least one marker light image frommarker light generated by at least one marker substance in the sample indifferent spectral ranges, and processing the sample and marker lightimages, wherein the imaging device comprises: (a) the light sourcedevice arranged to illuminate the sample under investigation withillumination light, said light source device including a narrowbandlight source arranged to excite a marker fluorescence from at least onemarker substance in the sample and a broadband light source arranged toilluminate the sample, (b) a detector device including at least onemulti-spectral sample light camera and at least one marker light cameraand being arranged to collect a plurality of images in response to saidillumination with the narrowband light source and the broadband lightsource, said plurality of images including: the at least one samplelight image reflected by the sample in at least one spectral range, andthe at least one marker light image originating from at least one markersubstance in the sample, wherein the at least one sample light camera isprovided with an optical filter which is arranged for passing visiblelight for adjusting the spectral range of the sample light backscatteredreflected by the sample, wherein a light splitting imaging optic isprovided which is configured for imaging light from the sample onto thecameras by relaying sample light onto the at least one sample lightcamera and marker light onto the at least one marker light camera sothat both of the sample light and the marker light are collectedsimultaneously, and (c) a processor adapted to process the at least onesample light image, and create at least one correction component fromthe at least one sample light image, the processor device furtheradapted to correct the marker light image using the at least onecorrection component.
 17. The imaging method according to claim 16,further comprising rendering at least one corrected marker light imagebased on the at least one sample light image and the at least one markerlight image.
 18. The imaging method according to claim 16, furthercomprising at least one of: rendering the at least one corrected markerlight image in a real-time mode, adjusting at least one of a spectralcharacteristic, a temporal characteristic, a polarization, a directionand a light field shape of the illumination light, adjusting a spectralor temporal sensitivity of each of sample light and marker lightcameras, and collecting an opto-acoustic image of the sample with anopto-acoustic imaging device.
 19. The imaging method according to claim16, wherein the sample includes at least one of: the sample comprisesbiological tissue, and the sample includes multiple marker substanceshaving different spectroscopic properties.
 20. The imaging methodaccording to claim 16, that performs medical imaging.
 21. The imagingmethod according to claim 16, wherein the medical imaging is associatedwith intra-operative, laparoscopic or endoscopic applications.